Radiohalogenated agents for in situ immune modulated cancer vaccination

ABSTRACT

A method of treating a malignant solid tumor in a subject is disclosed herein. The method includes the steps of administering to the subject an immunomodulatory dose of a radiohalogenated compound that is differentially taken up by and retained within malignant solid tumor tissue, and performing in situ tumor vaccination in the subject by intratumorally injecting into (or treating via a separate method) at least one of the malignant solid tumors a composition that includes one or more agents capable of stimulating specific immune cells within the tumor microenvironment. In certain exemplary embodiments, the radiohalogenated compound has the formula: 
                         
wherein R 1  is a radioactive halogen isotope, n is  18  and R 2  is —N + (CH 3 ) 3 .

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional Application No.62/363,608 filed on Jul. 18, 2016, which is incorporated by referenceherein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under CA197078 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD OF THE DISCLOSURE

This disclosure relates generally to methods of treating cancer. Inparticular, the disclosure is directed to methods of treating a cancercomprising one or more malignant solid tumors in a subject by (a)systemically administering to the subject an immunomodulatory dose of aradioiodinated compound that is differentially taken up by and retainedwithin solid tumor tissue, and (b) performing in situ tumor vaccinationin the subject at one of the malignant solid tumors using one or moretreatments capable of stimulating specific immune cells within the tumormicroenvironment.

BACKGROUND

Current cancer treatment typically involves systemic chemotherapywhereby non-targeted small molecule or antibody directed cytotoxicagents preferentially enter, or bind to (in the case of antibodydirected agents) and kill cancer cells by a variety of mechanisms.External beam radiation therapy (xRT), which is often combined withchemotherapy, kills cancer cells by inducing nuclear DNA double strandbreaks resulting in cell-cycle death. Unlike systemic chemotherapy, xRTdepends on the ability to accurately determine the anatomic location ofthe tumor. Surgical resection of tumors also depends on the ability tosee the tumor and on complete removal, since residual tumor cells willquickly reestablish the tumor following surgery. Surgery and xRT aregenerally limited to the local treatment of malignant tumors and thusare limited in treating disseminated or metastatic disease, which is whychemotherapy is often used in conjunction with these treatmentmodalities. Although systemic chemotherapy is capable of reaching manydistant metastatic sites, with the possible exception of brainmetastases, for all too many patients, responses are typicallyshort-lived (months to several years) and ultimately result in tumorrecurrence.

Because the body's natural immune system is also capable of destroyingcancer cells following their recognition, immunologic approaches arerapidly becoming more prevalent in cancer treatment paradigms. However,some cancer cells, and to a greater extent cancer stem cells, manage toinitially avoid immune-surveillance and actually acquire the ability toevolve and ultimately survive by remaining relatively immune invisible[Gaipi et al, Immunotherapy 6:597-610, 2014].

One specific immunologic approach that is being increasinglyinvestigated is “in situ vaccination,” a strategy that seeks to enhancetumor immunogenicity, generate tumor infiltrating lymphocytes (TIL) anddrive a systemic anti-tumor immune response directed against“unvaccinated,” disseminated tumors. In in situ vaccination, a malignantsolid tumor is injected with (or treated with) one or more agents thatfacilitate the release of tumor antigens while simultaneously providingpro-inflammatory signals to reverse the immune-tolerizingmicroenvironment of the tumor [Pierce et al, Human Vaccines &immunotherapeutics 11(8): 1901-1909, 2015; Marabelle et al, Clin. CancerRes. 20(7):1747-56, 2014; Morris et al, Cancer Research, e-pub ahead ofprint, 2016]. Although recent data from clinical trials and pre-clinicalmodels illustrate the potential of such an approach, there is a greatneed in the art for in-situr vaccination methods exhibiting improvedsystemic efficacy.

Radiation hormesis is a decades-old hypothesis that low doses ofionizing RT can be beneficial by stimulating the activation of naturalprotective repair mechanisms that are not activated in the absence ofionizing RT [Cameron and Moulder, Med. Phys. 25:1407, 1998]. The reserverepair mechanisms are hypothesized to be sufficiently effective whenstimulated as to not only cancel the detrimental effects of ionizing RTbut also inhibit disease not related to RT exposure. Perhaps related,the abscopal effect is a phenomenon reported in the 1950's, whereby, xRTtreatment of one tumor actually causes shrinkage of another tumoroutside the RT treatment area. Although rare, this phenomenon is thoughtto be dependent on activation of the immune system. Together, hormesisand the abscopal effect support the potential interaction andstimulation of the immune system by low dosage (immune stimulatory butnon-cytotoxic) RT, which may then be combined with other immunologicapproaches, such as in situ vaccination.

We have previously published that the combination of local xRT+in situvaccination are potently synergistic in treating large establishedtumors in mice, when there is a single tumor present [Morris et al,Cancer Research, e-pub ahead of print, 2016]. We have surprisinglydiscovered (and disclose herein) that the combination of in situvaccination and xRT does not result in inhibited tumor growth in thepresence of a second, non-radiated tumor. Apparently, the non-radiatedtumor exhibits a dampening effect (which we have designated as“concomitant immune tolerance”) on the immunomodulatory effect of thexRT and in situ vaccine on the radiated tumor. This concomitant immunetolerance can be overcome, enabling efficacy of in situ vaccination,when xRT is given to all areas of tumor. However, xRT cannot beeffectively used in combination with in situ vaccination methods in thepresence of multiple tumors, particularly if the tumors are not few innumber, or if the location of one or more of the tumors is not preciselyknown, or if it is not feasible to deliver xRT to all sites of tumor.Accordingly, in combination with in situ vaccination, there is a needfor improved methods of delivering an immunomodulatory dose of RT to alltumors within a subject, regardless of their number and anatomiclocation.

BRIEF SUMMARY

We have previously shown that certain alkylphosphocholine analogs arepreferentially taken up and retained by malignant solid tumor cells. InU.S. Patent Publication No. 2014/0030187, which is incorporated byreference herein in its entirety, Weichert et al. disclose using analogsof the base compound 18-(p-iodophenyl)octadecyl phosphocholine (NM404;see FIG. 1) for detecting and locating, as well as for treating, avariety of malignant solid tumors. If the iodo moiety is animaging-optimized radionuclide, such as iodine-124 ([¹²⁴I]-NM404), theanalog can be used in positron emission tomography-computed tomography(PET/CT) or single-photon emission computed tomography (SPECT) imagingof solid tumors. Alternatively, if the iodo moiety is a radionuclideoptimized for delivering therapeutic doses of RT to the solid tumorscells in which the analog is taken up, such as iodine-125 or iodine-131([¹²⁵I]-NM404 or [¹³¹I]-NM404), the analog can be used to treat thesolid tumors.

Such analogs not only target a wide variety of solid tumor types invivo, but also undergo prolonged selective retention in tumor cells,thus affording high potential as radiotherapy agents. Moreover, tumoruptake is limited to malignant cancer and not premalignant or benignlesions. Thus, such agents are well suited for delivering asub-cytotoxic but immunomodulatory dose of ionizing RT to all malignanttumors present within a subject, regardless of whether their number andlocations are known.

Accordingly, in a first aspect, the disclosure encompasses a method oftreating a cancer comprising one or more malignant solid tumors in asubject. The method includes the steps of: (a) administering to thesubject an immunomodulatory dose of a radiohalogenated compound that isdifferentially taken up by and retained within malignant solid tumortissue; and (b) performing in situ tumor vaccination in the subject atone or more of the malignant solid tumors using one or more treatmentscapable of stimulating specific immune cells within the tumormicroenvironment. An “immunomodulatory dose” is a low or sub-cytotoxicRT dose of the targeted radiotherapy agent. Although NM404 is used inthe examples below, the targeted radiotherapy agent could be anytargeted radiohalogenated therapy agent that include alpha, beta, auger,and/or gamma emitters, including, without limitation, radioiodinatedmetaiodobenzylguanidine (MIBG). The key feature is that targetedradiotherapy agent emits low or sub-cytotoxic RT doses that are notlethal to either the cancer cells or the relevant immune cells.

In some embodiments, the one or more treatments capable of stimulatingspecific immune cells can include xRT. In some embodiments, the one ormore treatments capable of stimulating specific immune cells includeintratumorally injecting into at least one of the malignant solid tumorsa composition that includes one or more agents capable of stimulatingspecific immune cells within the tumor microenvironment. In someembodiments, the one or more agents capable of stimulating specificimmune cells can include an immunostimulatory monoclonal antibody (mAb),a pattern recognition receptor agonist, an immunostimulatory cytokine,an immune stimulatory nanoparticle, an oncolytic virus, or anycombinations thereof. Non-limiting examples of immunostimulatorymonoclonal antibodies that could be used include anti-GD2 antibodies,anti-CTLA-4 antibodies, anti-CD137 antibodies, anti-CD134 antibodies,anti-PD-1 antibodies, anti-KIR antibodies, anti-LAG-3 antibodies,anti-PD-L1 antibodies, anti-CD40 antibodies, or combinations thereof. Insome embodiments, the immunostimulatory mAb is an antibody to atumor-specific antigen. In some embodiments, the composition thatincludes one or more immunostimulatory monoclonal antibodies may alsoinclude interleukin-2 (IL-2). In some embodiments, the anti-GD2 mAb thatis used may include hu14.18, and optionally, may further include IL-2(i.e., a fusion protein of the two).

In some embodiments, the immunostimulatory cytokine is IL-2,interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-21 (IL-21),or an interferon (IFN).

In some embodiments, the pattern recognition receptor agonist is anagonist of a toll-like receptor (TLR). Non-limiting examples of suchTLRs TLR include TLR-1, TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7, TLR-8,TLR-9, or TLR-10.

In some embodiments, the radiohalogenated compound ismetaiodobenzylguanidine (MIBG), wherein the iodine atom is a radioactiveiodine isotope. In some embodiments, the radioactive iodine isotope is¹²³I, ¹²⁴I, ¹²⁵I or ¹³¹I.

In some embodiments, the radiohalogenated compound has the formula:

or a salt thereof. R₁ is or includes a radioactive halogenated isotope,a is 0 or 1, n is an integer from 12 to 30, m is 0 or 1, Y is —H, —OH,—COOH, —COOX, —OX, or —OCOX, wherein X is an alkyl or an arylalkyl, andR₂ is —N⁺H₃, —N⁺H₂Z, —N⁺HZ₂, or —N⁺Z₃, wherein each Z is independentlyan alkyl or an aryl.

In some embodiments, the radioactive halogen isotope is a radioactiveisotope of iodine, bromine, or astatine. In some such embodiments, theradioactive halogen isotope is ²¹¹I, ¹²³I, ¹²⁴I, ¹²⁵I or ¹³¹I. In somesuch embodiments, the radioactive halogen isotope is ¹²⁵I or ¹³¹I. Insome such embodiments, the radioactive halogen isotope is ¹³¹I.

In some embodiments, a is 1 and m is 0. In some embodiments, n is 18. Insome embodiments, R₂ is —N⁺H₃.

In some embodiments, a is 1, m is 0, n is 18, and R₂ is —N⁺H₃. In somesuch embodiments, the radioactive halogen isotope is ²¹¹As, ¹²³I, ¹²⁴I,¹²⁵I, or ¹³¹I (the compound is [²¹¹As]-NM404, [¹²³I]-NM404,[¹²⁴I]-NM404, [¹²⁵I]-NM404 or [¹³¹I]-NM404).

In some embodiments, the radio-halogenated compound is administeredintravenously.

In some embodiments, the subject is a human.

In some embodiments, the method optionally includes the step of exposingone of the malignant solid tumors to xRT.

In some embodiments, the method optionally includes the step ofdetermining the immunomodulatory dose of the radiohalogenated compound.In some such embodiments, this step is performed by administering to thesubject a detection-facilitating dose of the radiohalogenated compound,and subsequently detecting signals originating from the one or moremalignant solid tumors within the subject that are characteristic of theradioactive halogen isotope within the radiohalogenated compound. Insome such embodiments, the radioactive halogen isotope contained in theradiohalogenated compound used in the detection-facilitating dose is²¹¹As, ¹²³I, ¹²⁴I, ¹²⁵I, or ¹³¹I. A non-limiting exemplaryradiohalogenated compound that could be used is [¹²⁴I]-NM404. In somesuch embodiments, the immunomodulatory dose of the radiohalogenatedcompound is calculated from the strength of the signals originating fromthe one or more malignant solid tumors within the subject. Optionally,the step of detecting signals characteristic of the radioactive halogenisotope is performed by positron emission tomography (PET) imaging orsingle-photon emission computed tomography (SPECT) imaging.

Non-limiting examples of the cancers presenting as malignant solidtumors that could treated using the disclosed method include melanoma,neuroblastoma, lung cancer, adrenal cancer, colon cancer, colorectalcancer, ovarian cancer, prostate cancer, liver cancer, subcutaneouscancer, squamous cell of the skin or head and neck, intestinal cancer,cervical cancer, glioma, breast cancer, pancreatic cancer, soft tissuesarcomas, Ewings sarcoma, rhabdomyosarcoma, osteosarcoma,retinoblastoma, Wilms' tumor, and pediatric brain tumors.

Other objects, features and advantages of the present invention willbecome apparent after review of the specification, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structure of the base compound18-(p-iodophenyl)octadecyl phosphocholine (NM404).

FIGS. 2A, 2B and 2C are a series of graphs showing that xRT+IT-ICelicits in situ tumor vaccination. 2A) Tumor growth curves and 2B)Kaplan-Meier survival curves show synergy between xRT andIT-hu14.18-IL2. 71% (22/31) of mice treated with xRT+IT-IC are rendereddisease-free. 2C) 90% of these reject subsequent engraftment with B78melanoma.

FIG. 3 is a graph demonstrating concomitant immune tolerance. Primarytumor response is shown. A distant un-treated tumor suppresses responseto xRT+IT-IC in a 2-tumor B78 melanoma model, and this suppression canbe overcome be radiating the second tumor.

FIG. 4 is a graph showing that concomitant immune tolerance is due toTregs. Primary tumor response is shown. A distant un-treated tumorsuppresses response to xRT+IT-IC in a 2-tumor B78 melanoma model andthis suppression can be overcome by depleting Tregs (using transgenicDEREG mice that express diphtheria toxin receptors on their Tregs, andthus depleting Tregs by administering diphtheria toxin).

FIG. 5 is an image showing selective uptake of ¹²⁴I-NM404 by B78melanoma. A mouse bearing a ˜200 mm³ B78 tumor received IV ¹²⁴INM404 andhad serial PET/CT scans done. This image at 71 h shows selective uptakeby the tumor with some residual background uptake by the heart andliver.

FIG. 6 is a graph demonstrating that in situ vaccination can be elicitedin the presence of residual levels of molecular targeted radiationtherapy (TRT). Treatment with combined xRT+IT-IC is equally effective inthe presence or absence of 3 μCi 131I-NM404. This approximates theresidual activity of TRT that will be present when we deliver xRT (d0)followed by IT-IC (d6-10), as described in Example 4.

FIGS. 7A, 7B, 7C, 7D and 7E are a series of graphs showingtumor-specific inhibition of primary tumor response to the combinationof local RT+IT-IC by a distant untreated tumor in murine melanoma andpancreatic tumor models. C57BL/6 mice bearing a syngeneic,disialoganglioside-expressing (GD2+), primary flank tumor +/−a secondarytumor on the contralateral flank were treated to the primary tumor only,as indicated, with xRT on day “1” and intra-tumor (IT) injection of 50mcg of the anti-GD2 immunocytokine (IC), hu14.18-IL2 (a fusion ofanti-GD2 mAb and IL2), on day 6-10. Mean primary tumor volumes aredisplayed in FIGS. 7A and 7C-7E. 7A). In mice bearing a primary B78melanoma tumor, the presence of an untreated secondary B78 tumorantagonized primary tumor response to RT+IT-IC. We describe this effectas “concomitant immune tolerance”—an antagonistic effect of anon-treated distant tumor on the local response of a treated tumor toxRT+IT-IC. 7B) Kaplan-Meier survival curves are shown for mice in panelA plus replicate experiments. Nearly all mice were euthanized due toprimary tumor progression. 7C) In mice bearing a primary Panc02-GD2+pancreatic tumor, with or without a secondary Panc02-GD2− tumor on theopposite flank, the presence of an untreated Panc02 secondary tumorsuppressed the response of a primary Panc02-GD2+ tumor to RT+IT-IC. 7D)In mice bearing a primary B78 melanoma tumor, a secondary B78 tumorsuppressed primary tumor response to RT+IT-IC but a secondaryPanc02-GD2+ pancreatic tumor did not exert this effect. 7E) In micebearing a primary Panc02-GD2+ tumor a secondary Panc02-GD2− tumorsuppressed primary tumor response to combined xRT and IT-hu14.18-IL2,while a B78 secondary tumor did not. n=number of mice per group.NS=non-significant, ***p<0.001.

FIGS. 8A, 8B and 8C include immunohistochemistry images and graphsshowing that concomitant immune tolerance is circumvented by specificdepletion of regulator T cells (Tregs). 8A). Immunohistochemistry forthe Treg marker, FoxP3 (representative 400× images are shown) for tumorsevaluated on day 6 after xRT in mice with one (A1 and A2) or two (A3 andA4) tumors. Mice received no xRT, or xRT only to the primary tumor. Theprimary tumor is shown in A1-A3 and the secondary is shown in A4. Smallarrows point out some of the FoxP3+ cells (brown nuclei=FoxP3+,blue=hematoxylin counterstain). The graphs on the right display blindedquantification of FoxP3+ cells per 200× field, corresponding to theconditions shown in A1, A2, A3 and A4, respectively. 8B and 8C) DEREGmice express diphtheria toxin receptor under control of theTreg-specific FoxP3 promoter, enabling specific depletion of Tregs uponIP injection of diphtheria toxin. DEREG mice bearing primary andsecondary B78 melanoma tumors were treated with xRT+IT-IC to the primarytumor and IP injection of either diphtheria toxin or PBS (the first ofreplicate experiments are shown). Concomitant immune tolerance iseliminated following depletion of Tregs in these mice, resulting inimproved 8B) primary and 8C) secondary tumor response. n=number of miceper group. **p<0.01, ***p<0.001.

FIGS. 9A and 9B are graphs showing that concomitant immune tolerance isovercome by delivering xRT to both tumor sites. In mice bearing primaryand secondary B78 tumors, the secondary tumor suppresses primary tumorresponse to primary tumor treatment with xRT+IT-IC. This is overcome bydelivering 12 Gy xRT to both the primary and secondary tumors and IT-ICto the primary tumor, resulting in improved 9A) primary tumor response(the first of replicate experiments is shown) and 9B) aggregate animalsurvival from replicate experiments. n=number of mice per group.**p<0.01, ***p<0.001.

FIGS. 10A, 10B and 10C are a series of graphs showing that low dose xRTalone does not elicit in situ vaccination but does overcome concomitantimmune tolerance when delivered to distant tumor sites together with 12Gy+IT-IC treatment of an in situ vaccine site. 10A) In mice bearing aprimary B78 tumor only, 12 Gy+IT-IC elicits in situ vaccination (asshown previously) and results in complete tumor regression in most mice(4/6 in this experiment) and a memory immune response (Morris, CancerRes, 2016). On the other hand no animals exhibit complete tumorregression following either IT-IC alone or low dose (2 Gy) xRT+IT-IC(0/6 in both groups) p<0.05. 10B) In mice bearing a primary andsecondary B78 melanoma tumor, low dose xRT (2 Gy or 5 Gy) delivered tothe secondary tumor is comparable to 12 Gy in its capacity to overcomeconcomitant immune tolerance at the primary tumor. 10C) In these sameanimals, it is apparent that overcoming concomitant immune tolerance bydelivery of low dose xRT to the secondary tumor rescues a systemicresponse to IT-IC immunotherapy. In this context, when xRT is deliveredto all tumor sites then IT-IC injection of the primary tumor triggers asystemic anti-tumor effect that renders secondary tumor response to 2 Gyor 5 Gy greater than the response to 12 Gy xRT in absence of primarytumor IT-IC injection.

FIGS. 11A, 11B, 11C and 11D is a PET image (11A) and a series of bargraphs (11B, 11C and 11D) showing that low dose TRT with ¹³¹I-NM404effectively depletes tumor infiltrating FoxP3+ Tregs without systemicleukopenia or depletion of tumor infiltrating CD8+ effector T cells. Inmost clinical scenarios, it is not feasible to deliver external beam,even low dose, to all tumor sites without eliciting marked bone marrowdepletion and leukopenia that would result in immunosuppression. Here wetested whether TRT could be administered systemically to specificallydeplete tumor infiltrating suppressive immune cells (Tregs), withouttriggering systemic immune cell depletion and leukopenia. 11A) Dosimetrystudies in this B78 melanoma tumor model using positron-emitting¹²⁴I-NM404 confirm tumor-selective uptake of NM404. C57BL/6 mice bearingB78 tumors were treated with 60 μCi ¹³¹I-NM404. This activityapproximates the amount of ¹³¹I-NM404 necessary to deliver ˜2 Gy TRT toa B78 tumor. Peripheral blood and tumor samples were collected inuntreated control mice (C) and at 8 day intervals (T1=d8, T2=d16,T3=d24, T4=d32) thereafter. 11B) This dose of TRT did not result in anysignificant systemic leukopenia and 11C) did not significantly affectthe level of tumor infiltrating CD8+ effector T cells (ANOVA p=0.25).11D) However, tumor infiltrating FoxP3+ Tregs were significantlydepleted by this dose of TRT (ANOVA p=0.03; * p<0.05).

FIGS. 12A and 12B are graphs showing that low dose TRT with ¹³¹I-NM404effectively overcomes concomitant immune tolerance and rescues thesystemic anti-tumor effect of in situ vaccination. Given the capacity oflow dose ¹³¹I-NM404 TRT to deplete tumor-infiltrating Tregs withoutrendering a mouse leukopenic, we tested whether low dose ¹³¹I-NM404might effectively overcome concomitant immune tolerance. C57BL/6 micebearing two B78 tumors were treated with 60-mcCi ¹³¹I-NM404 on day 1(NM404), as indicated. After one half-life (day 8), animals received 12Gy xRT or no xRT to the primary tumor (in situ vaccine site). Controlmice receiving no ¹³¹I-NM404 were treated to the secondary tumor asindicated (0, 2, or 12 Gy). Mice received daily IT injections of IC tothe primary tumor (in situ vaccine site), as indicated, on days 13-17.12A) Primary tumor and 12B) secondary tumor response is shown anddemonstrates that administration of low dose TRT effectively overcomesconcomitant immune tolerance and rescues the systemic anti-tumor effectof in situ vaccination.

DETAILED DESCRIPTION I. In General

It is understood that this disclosure is not limited to the particularmethodology, protocols, materials, and reagents described, as these mayvary. The terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which will be limited only by any later-filednonprovisional applications.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. As well, the terms “a” (or “an”), “one or more” and “at leastone” can be used interchangeably herein. The terms “comprising” andvariations thereof do not have a limiting meaning where these termsappear in the description and claims. Accordingly, the terms“comprising”, “including”, and “having” can be used interchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications and patentsspecifically mentioned herein are incorporated by reference for allpurposes including describing and disclosing the chemicals, instruments,statistical analysis and methodologies which are reported in thepublications which might be used in connection with the invention. Allreferences cited in this specification are to be taken as indicative ofthe level of skill in the art.

The terminology as set forth herein is for description of theembodiments only and should not be construed as limiting of theinvention as a whole. Unless otherwise specified, “a,” “an,” “the,” and“at least one” are used interchangeably and mean one or more than one.

The disclosure is inclusive of the compounds described herein (includingintermediates) in any of their pharmaceutically acceptable forms,including isomers (e.g., diastereomers and enantiomers), tautomers,salts, solvates, polymorphs, prodrugs, and the like. In particular, if acompound is optically active, the invention specifically includes eachof the compound's enantiomers as well as racemic mixtures of theenantiomers. It should be understood that the term “compound” includesany or all of such forms, whether explicitly stated or not (although attimes, “salts” are explicitly stated).

“Pharmaceutically acceptable” as used herein means that the compound orcomposition or carrier is suitable for administration to a subject toachieve the treatments described herein, without unduly deleterious sideeffects in light of the necessity of the treatment.

The term “effective amount,” as used herein, refers to the amount of thecompounds or dosages that will elicit the biological or medical responseof a subject, tissue or cell that is being sought by the researcher,veterinarian, medical doctor or other clinician.

As used herein, “pharmaceutically-acceptable carrier” includes any andall dry powder, solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic agents, absorption delaying agents, and thelike. Pharmaceutically-acceptable carriers are materials, useful for thepurpose of administering the compounds in the method of the presentinvention, which are preferably non-toxic, and may be solid, liquid, orgaseous materials, which are otherwise inert and pharmaceuticallyacceptable, and are compatible with the compounds of the presentinvention. Examples of such carriers include, without limitation,various lactose, mannitol, oils such as corn oil, buffers such as PBS,saline, polyethylene glycol, glycerin, polypropylene glycol,dimethylsulfoxide, an amide such as dimethylacetamide, a protein such asalbumin, and a detergent such as Tween 80, mono- andoligopolysaccharides such as glucose, lactose, cyclodextrins and starch.

The term “administering” or “administration,” as used herein, refers toproviding the compound or pharmaceutical composition of the invention toa subject suffering from or at risk of the diseases or conditions to betreated or prevented.

A route of administration in pharmacology is the path by which a drug istaken into the body. Routes of administration may be generallyclassified by the location at which the substance is applied. Commonexamples may include oral and intravenous administration. Routes canalso be classified based on where the target of action is. Action may betopical (local), enteral (system-wide effect, but delivered through thegastrointestinal tract), or parenteral (systemic action, but deliveredby routes other than the GI tract), via lung by inhalation. One form oflocal administration refered to in this submission is intratumoral (IT),whereby an agent is injected directly into, or adjacent to, a knowntumor site.

A topical administration emphasizes local effect, and substance isapplied directly where its action is desired. Sometimes, however, theterm topical may be defined as applied to a localized area of the bodyor to the surface of a body part, without necessarily involving targeteffect of the substance, making the classification rather a variant ofthe classification based on application location. In an enteraladministration, the desired effect is systemic (non-local), substance isgiven via the digestive tract. In a parenteral administration, thedesired effect is systemic, and substance is given by routes other thanthe digestive tract.

Non-limiting examples for topical administrations may includeepicutaneous (application onto the skin), e.g., allergy testing ortypical local anesthesia, inhalational, e.g. asthma medications, enema,e.g., contrast media for imaging of the bowel, eye drops (onto theconjunctiva), e.g., antibiotics for conjunctivitis, ear drops, such asantibiotics and corticosteroids for otitis externa, and those throughmucous membranes in the body.

Enteral administration may be administration that involves any part ofthe gastrointestinal tract and has systemic effects. The examples mayinclude those by mouth (orally), many drugs as tablets, capsules, ordrops, those by gastric feeding tube, duodenal feeding tube, orgastrostomy, many drugs and enteral nutrition, and those rectally,various drugs in suppository.

Examples of parenteral administrations may include intravenous (into avein), e.g. many drugs, total parenteral nutrition intra-arterial (intoan artery), e.g., vasodilator drugs in the treatment of vasospasm andthrombolytic drugs for treatment of embolism, intraosseous infusion(into the bone marrow), intra-muscular, intracerebral (into the brainparenchyma), intracerebroventricular (into cerebral ventricular system),intrathecal (an injection into the spinal canal), and subcutaneous(under the skin). Among them, intraosseous infusion is, in effect, anindirect intravenous access because the bone marrow drains directly intothe venous system. Intraosseous infusion may be occasionally used fordrugs and fluids in emergency medicine and pediatrics when intravenousaccess is difficult.

The following abbreviations are used in this disclosure: ADCC, Antibodydependent cell-mediated cytotoxicity; B16, A melanoma syngeneic toC57Bl/6 mice; B78, A variant of B16 that expresses GD2, due totransfection with GD2 synthase; D, day; Hu14.18-IL2, The primaryimmunocytokine (reacts against GD2) used in the studies disclosed in theexamples; IC, Immunocytoline (a fusion protein of a tumor-reactive mAblinked to IL2); IL2, Interleukin 2; IT, Intratumoral; IV, Intravenous;mAb, Monoclonal antibody; MAHA, Mouse anti-human antibody; NM404, usedto designate the phospholipid ether shown in FIG. 1, which isselectively taken up by most tumors and used for TRT in the studiesdisclosed in the examples; NXS2, A neuroblastoma syngeneic to AJ mice;Panc02-GD2, A pancreatic cancer syngeneic to C57Bl/6 mice, expressingGD2, due to transfection with GD2 synthase; PLE, Phospho-lipid Ether;RT, Radiation therapy; TRT, Targeted radiotherapy; W, week; 9464D-GD2, Aneuroblastoma syngeneic to C57Bl/6 mice, expressing GD2, due totransfection with GD2 synthase.

II. The Invention

This disclosure is directed to methods of treating any cancer thatpresents as one or malignant solid tumors. The disclosed methods combinetwo treatment steps, with an unexpected synergy resulting in a muchimproved in situ vaccination effect against the malignant solid tumors.Specifically, an immunomodulatory dose of a radiohalogenated compoundthat is differentially taken up by and retained within malignant solidtumor tissue is administered to the patient, and in situ tumorvaccination is performed by intratumorally injecting into (or applyingto) at least one of the malignant solid tumors a composition thatincludes one or more agents capable of stimulating specific immune cellswithin the tumor microenvironment, either with or without additional xRTto at least one of the malignant solid tumors being treated withimmune-stimulating agents. The immunomodulatory dose of theradiohalogenated compound likely reduces Treg levels (and otherimmune-suppressive elements) and prevents the immune system dampening(concomitant immune tolerance) that occurs when xRT is used against atumor and one or more additional tumors are not radiated.

A. Intratumoral Immunization—In Situ Vaccination

Compositions used for intratumoral immunization may include, withoutlimitation, one or more cytokines, immune checkpoint inhibitors, patternrecognition agaonists, and/or immunostimulatory monoclonal antibodies,including antibodies against tumor-specific antigens. For a review ofintratumoral immunization/in situ vaccination strategies that are amongthose that could be used, see Pierce et al, Human Vaccines &lmmunotherapoeutics 11(8): 1901-1909, 2015; and Marabelle et al, Clin.Cancer Res. 20(7):1747-56, 2014; and Morris et al, Cancer Res., e-pubahead of print, 2016; all of which are incorporated by reference herein.In the non-limiting examples disclosed herein, imtratumoral immunizationwas performed by injecting a fusion protein of an anti-GD2 mAb andinterleukin 2 (hu14.18-IL2). However, the disclosed methods are not inany way limited by these examples.

B. Immunomodulatory Dose of a Radiohalogenated Compound

The radiohalogenated compound used must selectively target a wide rangeof solid tumor cell types, such that the RT emitted by theradiohalogenated compound is directed to malignant solid tumor tissuewithout substantially exposing other tissue types to the emitted RT.Radiohalogenated compounds having such characteristics include MIBG orthe phospholipid ether analogs disclosed herein. The radioactivehalogenated isotope included in the radiohalogenated compound may be anyradioactive halogen isotope known to emit ionizing RT in a form thatwould result in immunostimulation of the cells that take up thecompounds. In one non-limiting example, the incorporated radioactivehalogen isotope is a radioactive iodine isotope, such as iodine-131.

The immunomodulatory RT dose (as opposed to injected dose) of theradiohalogenated compound is much less than the dose that would be usedfor conventional RT therapy against malignant solid tumors.Specifically, the dose must be sufficient to stimulate a response inimmune cells within the tumor microenvironment (likely by reducingimmune-supressing Treg levels and other immunosuppressive cells ormolecules), while not ablating the desired immune cells that areresponsible for the in situ vaccine effect.

As noted in the examples, the proper immunomodulatory dose can becalculated from imaging data obtained after administering a“detection-facilitating” dose of a radiohalogenated compound. Thedetection-facilitating dose may be quite different than theimmunomodulatory dose, and the radioactive halogen isotope that isincorporated into the radiohalogenated compound may be different(although the rest of the compound structure should be the same). Theradioactive halogen isotope used in the detection step and dosimetrycalculations may be any radioactive halogen isotope known to emit RT ina form that is readily detectable by conventional imaging means.Non-limiting examples of “conventional imaging means” include gamma raydetection, PET scanning, and SPECT scanning. Non-limiting examples ofradioactive halogen isotopes that could be used include astatine-211,iodine-123, iodine-124, iodine-125, and iodine-131.

C. Methods of Synthesizing the Disclosed Analogs and Compositions

The alkylphosphocholine analogs used in the disclosed methods are knownin the art, as are methods of synthesizing such analogs. For detailsregarding synthetic materials and methods, see, e.g., U.S. PatentPublication Nos. 2010/0284929, 2010/0316567, 2012/0128596, 2014/0030187,and 2014/0023587, each of which is incorporated by reference herein inits entirety. Similarly, methods and compositions used in in situvaccination/intratumoral immunization cancer therapies are known in theart.

D. Application to a Wide Range of Adult and Pediatric Solid Tumors

As noted above, we have previously demonstrated that thealkylphosphocholine analogs used in the disclosed methods areselectively taken up in a wide range of adult and pediatric solidtumors, as confirmed by both in vivo imaging and tumor growth inhibitionstudies.

It is well-known in the art that the relative radiosensitivity of solidtumor cancer cell phenotypes ranges from those that have very low RTsensitivities (e.g., pancreas, colorectal, glioma and breast) to thosewith high RT sensitivities (e.g., lymphomas). A tumor with lowradiosensitivity is considered to be highly radioresistant and a highlyradiosensitive tumor is considered to have low radioresistance. Relativeradiosensitivity of cancer cells is commonly presented as the fractionthat survives 2-Gy of in vitro RT exposure (SF2). Cancers can becategorized or ranked by their relative radiosensitivity, and Table 1provides non-limiting examples of known SF2 values for some common solidtumors.

TABLE 1 Relative Radiosensitivity of Select Cancer Cell Types Tumor TypeCell Line SF₂ value Breast MDA-MB-231 0.82 Pancreatic Mia-Paca 0.80Colorectal HCT-29 0.75 Melanoma B-78 0.65 Glioma (brain) U-87 0.63 Lung(NSCLC) A-549 0.61 Prostate PC-3 0.55 Lymphoma EL-4 0.30 SF₂ = survivingfraction following exposure to 2 Gy of in vitro RT exposure * Severalcell lines

We have previously demonstrated good tumor uptake and growth inhibitionwith the disclosed compounds in a wide range of tumor types, includingboth highly radiosensitive tumors like lymphoma and also in highly RTresistant tumors like glioma, breast, pancreatic or colorectal tumors.Thus, quantitative imaging and dosimetry can be used without undueexperimentation to quantify the RT dose necessary to stimulate theimmune system in a wide variety of solid tumor types.

E. Dosage Forms and Administration Methods

In situ vaccination can be performed by intratumoral injection, butother administration can apply (topical or systemic). For thesynergistic targeted RT, any route of administration may be suitable. Inone embodiment, the disclosed alkylphosphocholine analogs may beadministered to the subject via intravenous injection. In anotherembodiment, the disclosed alkylphosphocholine analogs may beadministered to the subject via any other suitable systemic deliveries,such as parenteral, intranasal, sublingual, rectal, or transdermaladministrations.

In another embodiment, the disclosed alkylphosphocholine analogs may beadministered to the subject via nasal systems or mouth through, e.g.,inhalation.

In another embodiment, the disclosed alkylphosphocholine analogs may beadministered to the subject via intraperitoneal injection or IPinjection.

In certain embodiments, the disclosed alkylphosphocholine analogs may beprovided as pharmaceutically acceptable salts. Other salts may, however,be useful in the preparation of the alkylphosphocholine analogs or oftheir pharmaceutically acceptable salts. Suitable pharmaceuticallyacceptable salts include, without limitation, acid addition salts whichmay, for example, be formed by mixing a solution of thealkylphosphocholine analog with a solution of a pharmaceuticallyacceptable acid such as hydrochloric acid, sulphuric acid,methanesulphonic acid, fumaric acid, maleic acid, succinic acid, aceticacid, benzoic acid, oxalic acid, citric acid, tartaric acid, carbonicacid or phosphoric acid.

Where the disclosed alkylphosphocholine analogs have at least oneasymmetric center, they may accordingly exist as enantiomers. Where thedisclosed alkylphosphocholine analogs possess two or more asymmetriccenters, they may additionally exist as diastereoisomers. It is to beunderstood that all such isomers and mixtures thereof in any proportionare encompassed within the scope of the present disclosure.

The disclosure also includes methods of using pharmaceuticalcompositions comprising one or more of the disclosed alkylphosphocholineanalogs in association with a pharmaceutically acceptable carrier.Preferably these compositions are in unit dosage forms such as tablets,pills, capsules, powders, granules, sterile parenteral solutions orsuspensions, metered aerosol or liquid sprays, drops, ampoules,auto-injector devices or suppositories; for parenteral, intranasal,sublingual or rectal administration, or for administration by inhalationor insufflation.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutically acceptable carrier, e.g.conventional tableting ingredients such as corn starch, lactose,sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalciumphosphate or gums, and other pharmaceutical diluents, e.g. water, toform a solid preformulation composition containing a homogeneous mixturefor a compound of the present invention, or a pharmaceuticallyacceptable salt thereof. When referring to these preformulationcompositions as homogeneous, it is meant that the active ingredient isdispersed evenly throughout the composition so that the composition maybe easily subdivided into equally effective unit dosage forms such astablets, pills and capsules. This solid pre-formulation composition isthen subdivided into unit dosage forms of the type described abovecontaining from 0.1 to about 500 mg of the active ingredient of thepresent invention. Typical unit dosage forms contain from 1 to 100 mg,for example, 1, 2, 5, 10, 25, 50 or 100 mg, of the active ingredient.The tablets or pills of the novel composition can be coated or otherwisecompounded to provide a dosage affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich, serves to resist disintegration in the stomach and permits theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol and cellulose acetate.

The liquid forms in which the alkylphosphocholine analogs may beincorporated for administration orally or by injection include aqueoussolutions, suitably flavored syrups, aqueous or oil suspensions, andflavored emulsions with edible oils such as cottonseed oil, sesame oil,coconut oil or peanut oil, as well as elixirs and similar pharmaceuticalvehicles. Suitable dispersing or suspending agents for aqueoussuspensions include synthetic and natural gums such as tragacanth,acacia, alginate, dextran, sodium caboxymethylcellulose,methylcellulose, polyvinylpyrrolidone or gelatin.

The disclosed alkylphosphocholine analogs are particularly useful whenformulated in the form of a pharmaceutical injectable dosage, includingin combination with an injectable carrier system. As used herein,injectable and infusion dosage forms (i.e., parenteral dosage forms)include, but are not limited to, liposomal injectables or a lipidbilayer vesicle having phospholipids that encapsulate an active drugsubstance. Injection includes a sterile preparation intended forparenteral use.

Five distinct classes of injections exist as defined by the USP:emulsions, lipids, powders, solutions and suspensions. Emulsioninjection includes an emulsion comprising a sterile, pyrogen-freepreparation intended to be administered parenterally. Lipid complex andpowder for solution injection are sterile preparations intended forreconstitution to form a solution for parenteral use. Powder forsuspension injection is a sterile preparation intended forreconstitution to form a suspension for parenteral use. Powderlyophilized for liposomal suspension injection is a sterile freeze driedpreparation intended for reconstitution for parenteral use that isformulated in a manner allowing incorporation of liposomes, such as alipid bilayer vesicle having phospholipids used to encapsulate an activedrug substance within a lipid bilayer or in an aqueous space, wherebythe formulation may be formed upon reconstitution. Powder lyophilizedfor solution injection is a dosage form intended for the solutionprepared by lyophilization (“freeze drying”), whereby the processinvolves removing water from products in a frozen state at extremely lowpressures, and whereby subsequent addition of liquid creates a solutionthat conforms in all respects to the requirements for injections. Powderlyophilized for suspension injection is a liquid preparation intendedfor parenteral use that contains solids suspended in a suitable fluidmedium, and it conforms in all respects to the requirements for SterileSuspensions, whereby the medicinal agents intended for the suspensionare prepared by lyophilization. Solution injection involves a liquidpreparation containing one or more drug substances dissolved in asuitable solvent or mixture of mutually miscible solvents that issuitable for injection.

Solution concentrate injection involves a sterile preparation forparenteral use that, upon addition of suitable solvents, yields asolution conforming in all respects to the requirements for injections.Suspension injection involves a liquid preparation (suitable forinjection) containing solid particles dispersed throughout a liquidphase, whereby the particles are insoluble, and whereby an oil phase isdispersed throughout an aqueous phase or vice-versa. Suspensionliposomal injection is a liquid preparation (suitable for injection)having an oil phase dispersed throughout an aqueous phase in such amanner that liposomes (a lipid bilayer vesicle usually containingphospholipids used to encapsulate an active drug substance either withina lipid bilayer or in an aqueous space) are formed. Suspension sonicatedinjection is a liquid preparation (suitable for injection) containingsolid particles dispersed throughout a liquid phase, whereby theparticles are insoluble. In addition, the product may be sonicated as agas is bubbled through the suspension resulting in the formation ofmicrospheres by the solid particles.

The parenteral carrier system includes one or more pharmaceuticallysuitable excipients, such as solvents and co-solvents, solubilizingagents, wetting agents, suspending agents, thickening agents,emulsifying agents, chelating agents, buffers, pH adjusters,antioxidants, reducing agents, antimicrobial preservatives, bulkingagents, protectants, tonicity adjusters, and special additives.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and the following examples and fallwithin the scope of the appended claims.

III. Examples Introduction to the Examples

These examples demonstrate the potential of bringing together two verydistinct cutting-edge disciplines in cancer treatment research,capitalizing on an unexpected and very potent synergy. These disciplinesare: 1) systemically administered TRT and 2) locally-directed,antibody-mediated, cancer immunotherapy. The data presented hereinsuggest that powerful synergy results from combining these approaches.Together, these two strategies can be used to destroy visiblemacroscopic tumor in a way that enables the destroyed cancer cells tofunction as a potent in situ vaccine that creates tumor-specific T cellimmunity able to eradicate persistent residual metastatic disease, forany type of solid tumor in any location.

Our ongoing preclinical work has shown that combination oftumor-specific mAb with IL2 (to activate innate immune cells) results inaugmented antibody-dependent cell-mediated cytotoxicity (ADCC) [1,2]; aprocess that has already been translated into clinical benefit forchildren with neuroblastoma [3]. Recent preclinical data demonstratemore potent antitumor efficacy when the mAb-IL2 fusion protein isinjected intratumorally (IT) [4,5]. Remarkably, large tumors that do notrespond to these mAb/IL2 injections and continue growing if treated onlywith local xRT, can be completely eradicated if the xRT is combined withthe mAb/IL2 treatment. Most mice are cured and develop T cell memorythat rejects re-challenge with similar tumor cells [6]; demonstratingthat the combined xRT+mAb/IL2 is acting as a potent “in situ”anti-cancer vaccine.

A key limitation is that if there is another macroscopic tumor presentin these animals when they receive xRT+mAb/IL2 treatment to the primary(first) tumor, the second tumor will continue to grow and, to oursurprise, suppress the immune response, preventing any shrinkage of the1^(st) treated tumor. This “concomitant immune tolerance” results, inpart, from suppressive regulatory T cells (Tregs) in the 2^(nd) tumor.Delivering RT alone to both tumors has minimal anti-tumor effect, butdoes deplete these Tregs. Thus, when first tumors are treated withxRT+mAb/IL2, the addition of RT to the second tumor circumvents thisimmune tolerance, enabling eradication of both tumors [7]. Theseobservations indicate a limitation of in situ tumor vaccination in themetastatic setting, but also suggest a robust capacity of RT to overcomethis limitation.

xRT cannot typically be delivered to all metastatic sites withoutprohibitive normal tissue toxicity and immune suppression. Yet notdelivering xRT to all sites of macroscopic disease may leave inhibitoryimmune lineages intact, which are capable of suppressing the immunologicresponse to our local xRT+mAb/IL2 immunotherapy. What is needed,therefore, is a means to deliver RT to all tumor sites in a cancerpatient in a targeted manner.

We have developed TRT vehicles capable of targeting systemicallyadministered RT to both primary and metastatic cancers. One such TRTagent, ¹³¹I-NM404, An intravenously (IV) administered phospholipid ether(PLE) analog, has shown nearly universal tumor targeting properties inover 60 in vivo cancer and cancer stem cell models. This agent iscurrently being evaluated clinically in multiple imaging and therapytrials [8,9]. A systemic injection of ¹³¹I-NM404 localizes in all tumorsregardless of anatomic location and internally provide sufficient RT toablate intratumoral immunosuppressive pathways that can preventdevelopment of an effective, tumor-eradicating, immune response. Theunique attributes of this approach are the near universal tumortargeting capability of NM404, as well as the ability to deliverimmunomodulatory sub-lethal doses of RT to all tumor sites, somethingthat is not typically feasible with xRT. What is new about this is thatour TRT Agents may immuno-modulate all tumors regardless of anatomiclocation, overcoming concomitant tolerance, which will result in along-term in situ tumor vaccination effect following local xRT followedby injection of a tumor specific mAb+IL2. As an increasing number oftumor specific mAbs are becoming approved for clinical use, thiscombination strategy could be readily expanded to clinical applicationfor any tumor type that can be targeted by a tumor-reactive mAb.Furthermore, the approach can be readily generalized to all in situtumor vaccination strategies.

In these examples, we describe how to assess the ability of ¹³¹I-NM404and related analogs to initiate the systemic immunomodulatory responsenecessary to enable local combined xRT+mAb/IL2 treatment to induce apotent radioimmune-facilitated in situ cancer vaccine. A similarassessment could be done for combined PLE analog-delivered TRT withother in situ cancer vaccine methods.

In sum, we disclose herein the first effort to combine two differentmethods from seemingly disconnected cancer therapy discliplines into asingle unified treatment. The data presented in these examples indicatethat the two methods can be synergistically combined to effectivelyeliminate malignant solid tumors and to prevent tumor recurrence. Thethree key concepts underlying this approach are that (A) local xRT+ITmAb/IL2 eradicates an existing single tumor and generates T-cell memory(an in situ vaccine); (B) unless irradiated, distant tumors causeconcomitant immune tolerance, preventing in situ vaccine efficacy; and(C) unlike whole body RT, TRT can localize to all tumors, without severesystemic RT-induced immune suppression. These concepts, together withour data, lead to the conclusion that xRT+IT mAb/IL2 to a mouse'sprimary tumor, plus TRT to eliminate tolerance caused by metastases,will enable effective in situ vaccination to eradicate all malignantsolid tumor-based cancers (primary and metastatic sites).

In Example 1, we present background data from our B78 GD2+ model insupport of the claimed method.

In Example 2, we provide guidance for determining the dose of xRT neededfor optimal in situ vaccine effect to a primary tumor, and the lowestdose of xRT to a distant tumor needed to prevent concomitant immunetolerance.

In Example 3, we provide guidance for determining the ¹³¹I-NM404 dosingthat approximates the required dosing of xRT to metastases, asdetermined in Example 2, and subsequently evaluating the effects of that¹³¹I-NM404 dose on in vivo immune function.

In Example 4, we provide guidance for using data from Examples 2 and 3to design/test/develop a regimen of ¹³¹I-NM404+local xRT+IT-mAb/IL2 inmice bearing two or more tumors in order to destroy the locally treatedtumors and induce T-cell mediated eradication of all distant tumors.Critical issues of TRT and xRT dose and time are optimized for antitumorefficacy.

In Examples 5, 6, 7 and 8, we provide information and specific data fromexperimental studies performed in accordance with the guidance ofExamples 1-4.

Example 1: Background Supporting Data

The Sondel lab has shown that tumor-specific mAb+IL2 activates innateimmune cells to mediate ADCC in mice [2], with clinical benefit forchildren with neuroblastoma [3]. In mice, IV administration of thehu14.18-IL2 IC is more potent than IV administration of anti-GD2 mAb+IL2[2, 10]. This can provide dramatic antitumor effects against very smallrecently established GD2+ tumors or very small microscopic metastases,potentially accounting for the clinical use of this approach in patientsin remission but at great risk for relapse [3]. More potent antitumorefficacy against measurable, macroscopic tumors [i.e. ˜50 mm³ GD2+tumors] can be achieved when the IC is injected intratumorally (IT-IC)rather than IV [4,5].

We are now focusing on ways to provide benefit in the setting of muchlarger, macroscopic tumors. Mice bearing a moderately large (200 mm³)B78 melanoma tumor, established five weeks earlier, show no response toIV-IC, and are slowed in their growth by IT-IC, but the tumors continueto grow. These same 200 mm³ tumors also grow after 12 Gy of xRT. Incontrast, when the IT-IC and xRT are combined, 73% of the animals becometumor-free and appear cured of their disease (FIGS. 2A and 2B). Thesemice then show T-cell mediated rejection of rechallenge with the sametumor (FIG. 2C). Thus IT-IC+xRT synergize, inducing the tumor to becomean “in situ tumor vaccine” [6].

In order to simulate clinical metastases, we inoculate mice with B78 inone flank on d-1, and the other flank at week 2. At week 5, the firsttumor is 200 mm³, and the second is 50 mm³. We anticipated thatxRT+IT-IC would destroy the first tumor and that the resultant T cellresponse would then destroy the second. However, adding IT-IC to the xRThad virtually no effect on either the 50 mm³ tumor or the 200 mm³ tumor(FIG. 3). This demonstrated a key limitation to the therapy wedelivered; namely, if there is another tumor present when these mocereceive xRT+IT-IC to the first tumor, the second tumor will cause asystemic tumor-specific concomitant immune tolerance effect, preventingany shrinkage of either tumor. Importantly, we have found that local xRT(12 Gy) to the first and second tumor simultaneously, abrogates thistolerance effect, allowing IT-IC to the first tumor to induce an immuneresponse that eradicates both tumors in most mice (FIG. 4) [7]. Recentdata, using a Treg depleting mAb (not shown) or transgenic mice thatallow selective Treg depletion (FIG. 4) [7], demonstrate that thisimmune tolerance is mediated, in part, by regulatory T cells (Tregs); RTto the first and second tumors partially deplete these Tregs,potentially explaining how irradiating both tumors circumvents thetolerance effect [7].

While local xRT to both the first and second tumors circumventstolerance, clinical metastatic disease is often in several locations.All macroscopic metastatic disease must receive RT to block immunetolerance and enable xRT+IT-IC to effectively eradicate all tumor sites.However, delivery of 12 Gy xRT to all sites of disease may be akin to“total body RT” with major dose-dependent (potentially lethal) toxicityand profound systemic immune suppression.

Previously, the Weichert lab has pioneered the development of TRT, inorder to deliver RT to all systemic tumor sites, while mimimizing“off-target” RT to normal tissue (especially marrow and immune tissue).

Based on the finding that tumor cells contain an overabundance ofphospholipid ethers (PLE) [11], we synthesized over thirtyradioiodinated PLE analogs in hopes of identifying analogs that wouldselectively target tumors [12]. One of these, NM404, not only displayednear universal tumor uptake in all but three of over 70 in vivo modelsexamined regardless of anatomic location, including brain metastases andcancer stem cells, but also underwent prolonged selective retention onceit enetered tumor cells [8]. These diapeutic PLE analogs are unique inthat they avoid premalignant and inflammatory lesions. Surface membranelipid rafts, which are overexpressed on cancer cells relative to normalcells, serve as portals of entry for PLE's, including NM404, into cancerand cancer stem cells [8]. Radioiodinated NM404 (I-124 and I-131), whichhas now been evaluated in five phase 1 and 2 PET imaging trials andthree phase 1 TRT radiotherapy trials, respectively, affords similartumor uptake and retention properties in over a dozen human cancer types[8]. Excellent tumor uptake in the cancer models relevant to theseexamples (the B78 GD2+ murine melanoma) have been confirmed with¹²⁴I-NM404 PET imaging (FIG. 5).

Example 2: Determining Dosages of xRT

Our data suggest these four hypotheses: (1) the dose of xRT we have usedto treat a single tumor causes modest direct in vivo tumor death andincreases susceptibility to immune mediated death (via both ADCC and Tcells); (2) the strong T-cell response provided by the addition ofIT-IC, but not IT mAb, suggests that mAb binding to radiated tumorcells, in the presence of IL2, facilitates antigen presentation andaugmented induction of adaptive immunity; (3) the presence of a secondtumor prevents the xRT+IT-IC to the first tumor from causing virtuallyany anti-tumor effect, due to tolerance caused largely by the systemicactions of immunosuppressive cells present in the second tumor [such asTregs and possibly myeloid derived suppressor cells (MDSC)]; thistolerance can be circumvented by depletion of Tregs (FIG. 4) orirradiating the second tumor (FIG. 3); (4) the dose of RT needed at thesecond tumor to circumvent tolerance might be much lower than the xRTdose needed for the first tumor to become an “in situ vaccine” [14].

Optimizing xRT Dose for the Primary (“In Situ Vaccine”) Tumor Site.

Our in vivo studies of xRT+IT-IC have focused on one dose of 12 Gy tothe first tumor. This is based on our data showing that in vitro RTinduces a dose-dependent functional upregulation of Fas on B78 tumorcells (nearing peak at >12 Gy), coupled to our in vivo datademonstrating our in situ vaccine effect of xRT+IT-IC requires mice withfunctional Fas-L (6). We conducted in vivo pilot studies prior toselecting the 12 Gy dose, which showed higher dose (16 Gy) or increasedfractionation flank RT had toxicity (dermatitis, ulceration, and latelimb edema) and no improvement in tumor response. While we chose a 12 Gysingle fraction of xRT for our in vivo studies, as we move towardsclinical translation, it will be beneficial to better understand themechanism of the local xRT effect and its dose requirements, in order tosafely and effectively induce the in situ vaccine effect.

Our mouse data (FIGS. 2A, 2B and 2C) show that we can induce a potentvaccine effect with 12 Gy xRT+IT-IC, even though 12 Gy of xRT alonecauses no shrinkage of the tumor; it merely slows the progressivegrowth. We might see just as potent an in situ vaccine effect usinglower doses of RT. To test this, we will evaluate a range of xRT doses(from 4-16 Gy) as a single fraction in mice bearing a ˜200 mm³ B78tumor, followed by our standard IT-IC regimen (50 mcg/d on days 6-10).We will determine which xRT doses give optimal tumor eradication andT-cell memory, when combined with IT-IC. If doses lower than 12 Gy areless toxic and show comparable efficacy, such lower doses would bebetter targets for our xRT dose to the “in situ vaccine” site inExamples 3 and 4.

Optimizing xRT Dose at a Distant Tumor to Prevent Tolerance fromBlocking “In Situ Vaccination.”

Treating both the first and second tumors with 12 Gy (FIG. 3) enablesIT-IC to the first tumor to induce a potent response that eradicatesboth tumors. Our goal is to be able to accomplish this same in situvaccine effect by providing xRT+IT-IC to a single tumor while using theminimal RT dose necessary at metastatic sites to circumvent tolerance.We recognize that xRT itself, especially if widespread, can bemyelo/immunosuppressive. This is why we are pursuing TRT in Examples 3and 4. Even though it is targeted, TRT does have some systemic deliveryof RT. In order to minimize the systemic immune suppression from TRT, wewish to give as low of a dose of TRT as is needed to effectively inhibitthe tumor-induced immune tolerance, while not causing systemicRT-induced global immune suppression. Therefore, we will determine howlow of a dose of xRT needs to be delivered to the distant tumor in orderto enable a higher xRT dose to the first tumor to function as an in situvaccine when combined with IT-IC to the first tumor.

Mice bearing a 200 mm³ first B78 tumot and a ˜50 mm³ second B78 tumorwill receive 12 Gy of xRT to the first tumor on day-0 (˜5 weeks afterimplantation of the first B78 tumor). This will be followed by ourstandard regimen of IT-IC on days 6-10. Separate groups of mice willreceive varying doses of xRT to the second tumor. Based on data from thelab of B. Johnson demonstrating that a total body xRT of 3 Gy canprevent an immunosuppressive effect in a myeloma model (15), we willevaluate doses of 0, 1, 5 and 8 Gy (in addition to the 12 Gy dose weknow is effective). We will see if doses substantially less than 12 Gyto the second tumor can be as effective as the full 12 Gy dose ateliminating the immune tolerance.

Once we have clarified the critical dose of xRT where we lose thebeneficial effect, we will perform subsequent analyses to betteridentify the critical dose. For example, if 5 Gy is as effective as 12Gy, but 1 Gy is not much better than 0 Gy, we would then compare 2, 3,and 4 Gy to identify the critical lowest effective RT dose needed toeliminate tolerance and obtain efficacy in this two tumor model,receiving 12 Gy+IT-IC to the first tumor.

Repeat studies would then be done to confirm if this lowest effectivedose to the second tumor still enables an effective in situ vaccine whenthe dose to the first is the lowest effective dose in the 1-tumor model(tested in Example 2, above) rather than the 12 Gy dose. In summary, thestudies of Example 2 will determine what the lowest xRT doses are forthe first and second tumors, without losing the efficacy we havedemonstrated with 12 Gy to both.

Initiating Studies of Required xRT Dose to First and Second Tumors inMice Bearing Tumors Other than B78.

To allow our mouse studies to suggest more clinical generalizability, wewill initiate analyses of RT+IT-IC in additional models of GD2+ tumors.We have published on IT-IC with hu14.18-IL2 IC in AJ mice bearing theGD2+ NXS2 neuroblastoma [5]. We are also evaluating IT-IC with this sameIC in C57BL/6 mice bearing the GD2+9464D-GD2 neuroblastoma, and thePanc02-GD2 pancreatic cancer that express GD2 via our insertion of thegene for GD2 synthase. As for Example 2, for each model we willdetermine the lowest effective xRT dose needed to the primary and thesecondary tumors to retain the in situ vaccine effect.

Example 3

Determining Dosage of ³¹I-NM404 and Evaluating Effects on ImmuneFunction

Dosimetry with TRT and Immunesuppression from TRT in C57BL/6 Mice.

¹³¹I-NM404 has shown selective uptake in vitro in >95% of tumor lines(human and mouse), with poor uptake by non-malignant cells, and withsimilar tumor specificity seen in vivo. This includes selective uptakein vivo with the B78 tumor (FIG. 5). In our preliminary dosimetry study,we gave ¹²⁴I-NM404 to C57BL/6 mice and characterized the time course ofTRT exposure by serial PET/CT imaging (as in FIG. 5). Monte Carlodosimetry calculations [16-18] based on this study indicated that ˜60μCi of ¹³¹I-NM404 would be needed to deliver ˜3 Gy to an established B78tumor over a four week period of decay. After those four weeks, theremaining TRT dose to the B78 tumor would be less than 0.25 Gy. We willreplicate the data we obtained in our 2-tumor model using xRT (FIG. 3),but use the lowest possible dose of targeted ¹³¹I-NM404 TRT to enableeffective elimination of tumor-induced tolerance at all sites of distantdisease. However, unlike xRT, which delivers all dose in minutes and isthen done, TRT deposits dose over time, depending upon both thebiological and physical half-life of the targeted isotope (8 day t1/2for ¹³¹I). We want an initial TRT effect at the distant tumor sites toeradicate immune tolerance; however we want the immunosuppressive TRTeffect to then be minimal when we give the IT-IC to induce ADCC and thein situ vaccine anti-tumor effects. This is essential to allow fulltumor destruction at all sites.

Using the dosimetry calculations from our preliminary data, we estimatedthat a dose of 3 μCi of ¹³¹I-NM404, should deliver an equivalent of ˜0.2Gy to the tumor site, a dose that we hypothesized should not beimmunosuppressive and should not prevent lymphocyte-mediated tumordestruction. As noted above, this is the dose we estimated would remainyet to be delivered 28 days after an initial ¹³¹I-NM404 dose of 60 μCi.We thus evaluated groups of mice bearing a single 200 mm³ B78 tumor. Onday 0, all mice got 12 Gy xRT to their tumor, and on days 6-10, all got50 mcg/d of IT-IC. One group also got 3 μCi of ¹³¹I-NM404 on d-0 (˜0.2Gy). FIG. 6 shows that the group receiving the ¹³¹I-NM404 had the samedegree of tumor eradication as the group without ¹³¹I-NM404,demonstrating that this low dose of “residual” TRT in the tumor does notblock immune mediated destruction by the RT+IT-IC in situ vaccine. Wethus hypothesize that if we would use an initial dose of 60 μCi of¹³¹I-NM404 TRT on day-22, it would effectively block the tolerogeniceffect of distant tumors, yet enable xRT on day 0 and IT-IC on days 6-10(28 d after the TRT) to the first tumor to function as an in situvaccine, inducing an adaptive response that then eradicates all tumors.

The experiments outlined in this example will evaluate the doserelationships tested in FIG. 6. In our 1-tumor B78 model, we will test arange of doses of ¹³¹I-NM404 TRT to determine what TRT dose would resultin enough unwanted systemic immune suppression to interfere with thedesired in situ vaccine effect (and thereby slow or prevent eradicationof the first tumor). This is important to Example 4, as it will allow usto make sure the residual radioactivity of the TRT has decayed to lessthan this value at the time we initiate IT-IC to the first tumor in micewith distant disease. We will also evaluate the kinetics of the TRTresponse after varying TRT doses to determine how long we must waitafter the “tolerance-preventing TRT dose” is given to animals withmultiple tumors to allow the RT+IT-IC treatment of the first tumor tostill induce the in situ vaccine effect and eradicate the primary aswell as distant tumors.

Related studies will also look at what dose of TRT, given as singleagent treatment, would be required to cause slowing, versus shrinkage,versus eradication of a single B78 tumor. The dose of TRT that will beneeded to eliminate the tumor-induced immune tolerance will besubstantially less than the TRT dose needed to actually induce completetumor destruction (from the TRT alone).

Finally, once the effects of various doses of TRT are determined in the1-tumor model, we will evaluate the subtle immune-suppressive effects ofTRT, by evaluating sera from these mice for their immune response to thehuman IgG component of the IC. We have shown that immunocompetent micegenerate a readily quantified level of Mouse Anti-Human Antibody (MAHA)following treatment with these humanized ICs (19). We will use this as ameans of determining at what dose we are seeing the TRT cause adetectible dose-dependent decrease in the strength of the murine immuneresponse, to gauge the overall immunosuppressive effects from thesystemic doses of RT these mice will receive from this TRT. The low TRTdose that we will need to block the tumor-induced immune tolerance willcause minimal systemic immune suppression.

Dosimetry with TRT and Immunesuppression from TRT in A/J Mice.

As in Example 2, once we are nearing completion of the studies outlinedabove, we will begin initiating selective replicate studies in A/J micebearing the NXS2 neuroblastoma.

Example 4: Developing a Regimen of ¹³¹I-NM404+Local xRT+IT-mAb/IL2 inMice Bearing Two or More Tumors

Testing the Efficacy of TRT+xRT+IT-IC in the 2-Tumor B78 Model.

The dose and timing information learned from the studies outlined inExamples 2 and 3 will provide the information we need to estimate TRTdosing and timing required for efficacy in our 2-tumor model. C57BL/6mice will be inoculated with B78 in the left (L) and right (R) flankssimultaneously. Each tumor should be ˜50 mm³ after two weeks and ˜200mm³ after five weeks. If we assume that our dosimetry calculations inExample 3 suggest that we need to deliver 60 μCi of TRT to approximate 3Gy RT to the second tumor (to block the immune tolerance), our xRTstudies predict that this dose should have minimal slowing effect ontumor growth. We would plan to treat different groups of mice with 30,60 or 90 μCi at the 2 w time point (when the tumors are ˜50 mm³). Threeweeks later the tumors should be ˜200 mm³; at that time we will give xRT(dose determined as outlined in Example 2) followed six days later (˜28d after the TRT) by five daily injections of IT-IC to the tumor in the Lflank, to induce the in situ vaccine effect. Control mice would get noTRT, and only the xRT and IT-IC to the L flank, anticipating no in situvaccine due to tolerance from the distant tumor. A separate group wouldget local xRT to both tumors and IT-IC to the L flank, anticipatingeradication of both tumors via the in situ vaccine effect. Another groupwould get TRT+IT-IC, but without local xRT, anticipating an incompletevaccine effect.

Follow-up experiments would further evaluate varying doses of TRT andvariations in the timing between the TRT and the local xRT+IT-IC to theprimary tumor (L flank). The readouts will be: (A) eradication of theprimary tumor; (B) eradication of the secondary tumor; and (C) systemicimmune suppression, via ELISA analyses of the MAHA response. Our goal isto identify TRT dose and timing, to add to the local xRT+IT-IC regimenthat can eradicate both tumors in most mice, while minimizing systemicimmunosupression (as measured by MAHA response). We anticipate that wewill find conditions for TRT administration and timing that can becombined with local xRT+IT-IC to the primary tumor that are as effectiveas our regimen of IT-IC to the primary tumor following xRT to bothtumors (FIG. 3).

Testing the Efficacy of TRT+xRT+IT-IC in Mice Bearing More than Two B78Tumors.

This section of Example 4 is most analogous to the relevant clinicalsetting; namely, patients with an injectable tumor that could be used asan in situ vaccine site, but with multiple distant metastases that couldeach be causing tumor-induced immune tolerance. These studies willreplicate the conditions found to be most effective in the first part ofExample 4 (above). The important difference is that these mice will eachhave four separate tumors, in L and R flanks, and L and R para-scapularregions. The TRT would be given at the dose and timing found mosteffective in the studies outlined in the first section of Example 4,with xRT+IT-IC subsequently given only to the L-flank lesion. The goalhere is to test TRT dose and timing issues to enable an effective insitu vaccine to function, because the TRT would effectively eliminatethe tumor-induced immune tolerance caused by the three sites not gettingxRT. The measure of efficacy would be elimination of all four tumors inmost mice. Modifications in TRT dose and timing will be studied in orderto generate a regimen that is most effective. Such a regimen could thenbe considered for future translation to the clinic for patients withmultiple distant metastases, that could not all be irradiated viaexternal beam, but could be irradiated via TRT, when combined with localxRT+IT-IC to the “in situ vaccine” site.

Initiating Studies of TRT+xRT and IT-IC in Mice Bearing NXS2, 9464D-GD2or Panc02-GD2 in Two or More Sites.

As in Examples 2 and 3, once the studies outlined in the previoussections of Example 4 are progressing, we will initiate similar studiesin mice bearing NXS2, 9464D-GD2 or Panc02-GD2 in two or more sites.

Example 5: Experiments Determining the Dose of xRT Needed for Optimal InSitu Vaccine Effect to a Primary Tumor, and the Lowest Dose of xRT to aDistant Tumor Needed to Prevent Concomitant Immune Tolerance

Dose titration experiments, evaluating a variety of xRT doses, to micewith 1 or 2 tumors have been performed. The first goal has been to testthe dose of xRT needed in mice with one tumor to facilitate synergy andan “in situ vaccine” with IT-IC, tumor-reactive mAb linked to IL2.Initial experiments have confirmed our prior observation that 12 Gy RTalone does not eradicate or even regress the growth of established B78melanoma tumors (0% complete regression), whereas 12 Gy+IT-IC results incomplete regression of most B78 tumors (66%) in mice bearing a singletumor. On the other hand, 2 Gy+IT-IC slows tumor progression compared toIT-IC alone (mean tumor size day 32=472 mm³ vs 1214 mm³, respectively)but did not render any mice disease free (0% complete regression).

In our “2-tumor model”, we have previously shown that treatment of one“primary” tumor with xRT+IT-IC is not effective in treating either thetreated primary tumor or the untreated “secondary” tumor. In fact, inthis 2-tumor model we have observed that the presence of the secondtumor eliminates the efficacy of IT-IC injection following xRT. We havedesignated this phenomenon as “concomitant immune tolerance” (CIT), anddemonstrated that this results, at least in part, from T regulatorycells (Tregs) in the distant (non-irradiated) secondary tumor, whichcirculate systemically and repopulate the xRT-treated/IT-IC injectedprimary tumor. These Tregs that return to the primary tumor appear tointerfere with the desired “in situ vaccine” effect.

We have now confirmed our prior observation that CIT can be overcome bydelivering 12 Gy xRT to both the primary and the secondary tumor.Importantly, given that Tregs are quite sensitive to RT, we hypothesizedthat a lower dose of RT could be delivered to the secondary tumor inorder to overcome CIT and rescue response to in situ vaccination at theprimary tumor (primary tumor treated with 12 Gy+IT-IC). We have nowtested this and observed that xRT doses of 2 Gy or 5 Gy to the secondarytumor are comparable to 12 Gy in their capacity to blunt CIT and rescueresponse to primary tumor treatment with 12 Gy+IT-IC. These importantexperiments have been repeated in duplicate, and suggest (ashypothesized) that the dose of xRT that must be given to distant tumorsto prevent CIT is much less than the dose needed at the IT-IC injectedprimary tumor site for the purpose of generating an in situ vaccineeffect.

This supports our overarching hypothesis in this disclosure, andsuggests that in animals bearing multiple tumors we will be able todeliver a relatively low dose of RT to all sites of disease using thetargeted radiotherapeutic (TRT) NM404, and thereby overcome CIT whenthis is combined with local xRT and IT-IC injection of a single tumorsite (the in situ vaccine site).

Example 6: Experiments Determining the ¹³¹I-NM404 Dosing thatApproximates the Required Dosing of xRT to Metastases, as DeterminedAbove, and then Evaluating the Effects of that ¹³¹I-NM404 Dose on InVivo Immune Function

Based on the preliminary data described above, studies have been done tomove these concepts into in vivo testing using TRT. Dosimetry studieshave been performed on mice bearing 1 or 2 B78 tumors (the tumor modelthat we have used to demonstrate best our in situ vaccine approach andthe hurdle of CIT). This was done in order to estimate the amount of¹³¹I-NM404 that would be needed to approximate ˜2 Gy of xRT.

In order to then determine if a ˜2 Gy equivalent dose of ¹³¹I-NM404would have the desired effects against intratumor lymphoid cells(particularly Tregs), 2 separate approaches have been pursued. First, weadministered this dose of ¹³¹I-NM404 to mice bearing a radiosensitivelymphoma tumor, which exhibits comparable NM404 uptake to B78 tumors.Following this we have documented potent lymphoid tumorshrinkage/dose-dependent inhibition under conditions that did not causeeither substantial shrinkage/slowing of the B78 tumor or any evidentdepletion of circulating lymphoid cells (as gauged by peripheralcomplete blood counts). These data are consistent with the fact thatlymphoid cells are much more sensitive to low-dose RT than are typicalsolid tumor cells, and suggest that selective uptake of TRT in tumor mayenable intratumor lymphoid cell depletion without systemic lymphopenia.These studies also suggest that such a lymphoid tumor could serve as anin vivo biological “dosimeter” for identifying and monitoring the effectof TRT on intratumor lymphoid cells.

A second approach involved treating mice with B78 tumors with these samedoses of ¹³¹I-NM404. These animals were then sacrificed at half-life (8d) intervals, and after sufficient delay for radioactive decay, thetumors were stained for the presence of effector T cells and Tregs byimmunohistochemistry Intriguingly, the animals receiving ¹³¹I-NM404 inthis initial experiment showed no systemic lymphopenia at any time point(by peripheral complete blood count) but did show a decrease inintratumor FoxP3+ Tregs at 2 half-lifes following TRT administration. Atthis 2-half-life time point, we also observed a decrease in intratumoreffector CD8+ T cells. Importantly, however at subsequent 3 and 4half-life time points we observed an increase in intratumor CD8+effector T cells but a further decline in the levels of intratumorTregs, both compared to untreated baseline and 2^(nd) half-life levels.This observation again supports our hypothesis that it will be feasibleto use TRT to overcome Treg-mediated CIT in order to rescue an in situvaccine effect in animals bearing multiple tumors.

Finally, to characterizing the immunological effects of TRT on theimmune cells within tumors, we have treated B78 bearing mice with¹³¹I-NM404 and collected tumor tissue at pretreatment and at half-life(8 d) intervals thereafter. These tissues were then analyzed by RT-PCRfor gene expression of a panel of immune signatures. The resultsindicate that TRT treatment alone causes striking changes in expressionof tumor cell markers of immunsusceptibility and in genes normallyexpressed only by immune cells, with the latter showing a clear timecourse of decreased expression followed by rebound over-expression.

Example 7: Experiments Using Data from Examples 5 and 6 to Develop aRegimen of ¹³¹I-NM404+Local xRT+IT-mAb/IL2 in Mice Bearing Two or MoreTumors and Induce T-Cell Mediated Eradication of all Distant Tumors

This Example illustrates treating animals bearing tumors in at least 2locations. Our strategy involves using xRT and local IT-IC at the insitu vaccine site, in combination with TRT systemically to inhibit CIT,in order to obtain enhanced anti-tumor immune activity at all tumorsites. Critical issues of TRT and xRT dose and timing will be optimizedfor antitumor efficacy.

Using the data summarized in Examples 5 and 6, a study was done in micebearing 2 separate B78 tumors. Mice received the estimated requiredsystemic ¹³¹I-NM404 dose followed by xRT and local immunotherapy to thein situ vaccine site. With appropriate controls, this dose of ¹³¹I-NM404did appear to attenuate CIT, as desired in mice with 2 tumors. Inaddition, in mice with one tumor, this TRT dose did not appear tointerfere with the local in situ vaccine effect (as hypothesized anddesired). Further testing, and modification of some of the experimentalvariables, is underway in order to try to maximize the desired effect ofblocking CIT without suppressing the in situ vaccine effect. Moredetails regarding these experiments are disclosed in Example 8 below.

Example 8: Data from Mice Bearing Two or More Tumors

Tumor-Specific Inhibition of Primary Tumor Response to the Combinationof Local xRT+IT-IC by a Distant Untreated Tumor in Murine Melanoma andPancreatic Tumor Models.

C57BL/6 mice bearing a syngeneic, GD2+ primary flank tumor+/−a secondarytumor on the contralateral flank were treated to the primary tumor only,as indicated, with xRT on day “1” and IT injection of 50 mcg of theanti-GD2 IC, hu14.18-IL2 on day 6-10.

In mice bearing a primary B78 melanoma tumor, the presence of anuntreated secondary B78 tumor antagonized primary tumor response toxRT+IT-IC (FIG. 7A). We describe this effect as “concomitant immunetolerance”—an antagonistic effect of a non-treated distant tumor on thelocal response of a treated tumor to xRT+IT-IC. Kaplan-Meier survivalcurves were obtained for these mice plus replicate experiments (FIG.7B). Nearly all mice were euthanized due to primary tumor progression.

In mice bearing a primary Panc02-GD2+ pancreatic tumor, with or withouta secondary Panc02-GD2− tumor on the opposite flank, the presence of anuntreated Panc02 secondary tumor suppressed the response of a primaryPanc02-GD2+ tumor to xRT+IT-IC (FIG. 7C). In mice bearing a primary B78melanoma tumor, a secondary B78 tumor suppressed primary tumor responseto xRT+IT-IC but a secondary Panc02-GD2+ pancreatic tumor did not exertthis effect (FIG. 7D). In mice bearing a primary Panc02-GD2+ tumor asecondary Panc02-GD2− tumor suppressed primary tumor response tocombined xRT and IT-hu14.18-IL2, while a B78 secondary tumor did notFIG. 7E).

Concomitant Immune Tolerance is Circumvented by Specific Depletion ofRegulator T Cells (Tregs).

Immunohistochemistry images were obtained for the Treg marker, FoxP3 fortumors evaluated on day 6 after xRT in mice with one or two tumors (FIG.8A). Mice received no xRT, or xRT only to the primary tumor. DEREG miceexpress diphtheria toxin receptor under control of the Treg-specificFoxP3 promoter, enabling specific depletion of Tregs upon IP injectionof diphtheria toxin (FIGS. 8B and 8C). DEREG mice bearing primary andsecondary B78 melanoma tumors were treated with xRT+IT-IC to the primarytumor and IP injection of either diphtheria toxin or PBS. Concomitantimmune tolerance is eliminated following depletion of Tregs in thesemice, resulting in improved primary (FIG. 8B) and secondary (FIG. 8C)tumor response.

Concomitant Immune Tolerance is Overcome by Delivering xRT to Both TumorSites.

In mice bearing primary and secondary B78 tumors, the secondary tumorsuppresses primary tumor response to primary tumor treatment withxRT+IT-IC. This is overcome by delivering 12 Gy xRT to both the primaryand secondary tumors and IT-IC to the primary tumor, resulting inimproved primary tumor response (FIG. 9A) and aggregate animal survival(FIG. 9B) from replicate experiments.

Low Dose xRT Alone does not Elicit In Situ Vaccination but does OvercomeConcomitant Immune Tolerance when Delivered to Distant Tumor SitesTogether with 12 Gy+IT-IC Treatment of an In Situ Vaccine Site.

In mice bearing a primary B78 tumor only, 12 Gy+IT-IC elicits in situvaccination (as shown previously) and results in complete tumorregression in most mice (FIG. 10A) and a memory immune response (Morris,Cancer Res, 2016). On the other hand no animals exhibit complete tumorregression following either IT-IC alone or low dose (2 Gy) xRT+IT-IC(0/6 in both groups) p<0.05.

In mice bearing a primary and secondary B78 melanoma tumor, low dose xRT(2 Gy or 5 Gy) delivered to the secondary tumor is comparable to 12 Gyin its capacity to overcome concomitant immune tolerance at the primarytumor (FIG. 10B). In these same animals, it is apparent that overcomingconcomitant immune tolerance by delivery of low dose xRT to thesecondary tumor rescues a systemic response to IT-IC immunotherapy (FIG.10C). In this context, when RT is delivered to all tumor sites thenIT-IC injection of the primary tumor triggers a systemic anti-tumoreffect that renders secondary tumor response to 2 Gy or 5 Gy greaterthan the response to 12 Gy RT in absence of primary tumor IT-ICinjection.

Low Dose TRT with ¹³¹I-NM404 Effectively Depletes Tumor InfiltratingFoxP3+ Tregs without Systemic Leukopenia or Depletion of TumorInfiltrating CD8+ Effector T Cells.

In most clinical scenarios, it is not feasible to deliver external beam,even low dose, to all tumor sites without eliciting marked bone marrowdepletion and leukopenia that would result in immunosuppression. Here wetested whether TRT could be administered systemically to specificallydeplete tumor infiltrating suppressive immune cells (Tregs), withouttriggering systemic immune cell depletion and leukopenia. Dosimetrystudies in this B78 melanoma tumor model were performed usingpositron-emitting ¹²⁴I-NM404 confirm tumor-selective uptake of NM404(FIG. 11A). C57BL/6 mice bearing B78 tumors were treated with 60 μCi¹³¹I-NM404. This activity approximates the amount of ¹³¹I-NM404necessary to deliver ˜2 Gy TRT to a B78 tumor. Peripheral blood andtumor samples were collected in untreated control mice (C) and at 8 dayintervals (T1=d8, T2=d16, T3=d24, T4=d32) thereafter. This dose of TRTdid not result in any significant systemic leukopenia (FIG. 11B) and didnot significantly affect the level of tumor infiltrating CD8+ effector Tcells (FIG. 11C). However, tumor infiltrating FoxP3+ Tregs weresignificantly depleted by this dose of TRT (FIG. 11D).

Low Dose TRT with ¹³¹I-NM404 Effectively Overcomes Concomitant ImmuneTolerance and Rescues the Systemic Anti-Tumor Effect of In SituVaccination.

Given the capacity of low dose ¹³¹I-NM404 TRT to depletetumor-infiltrating Tregs without rendering a mouse leukopenic, we testedwhether low dose ¹³¹I-NM404 might effectively overcome concomitantimmune tolerance. C57BL/6 mice bearing two B78 tumors were treated with60-μCi¹³¹I-NM404 on day 1 (NM404), as indicated. After one half-life(day 8), animals received 12 Gy xRT or no xRT to the primary tumor (insitu vaccine site). Control mice receiving no ¹³¹I-NM404 were treated tothe secondary tumor as indicated (0, 2, or 12 Gy). Mice received dailyIT injections of IC to the primary tumor (in situ vaccine site), asindicated, on days 13-17. Primary tumor (FIG. 12A) and secondary tumor(FIG. 12B) response demonstrates that administration of low dose TRTeffectively overcomes concomitant immune tolerance and rescues thesystemic anti-tumor effect of in situ vaccination.

Conclusion to the Examples

These examples illustrate a novel, never before tested or considered,anti-cancer strategy, based on the synergistic and widely applicablecombination of two known therapeutic methods: (1) targeted systemicdelivery of radiotherapy (J. Weichert and colleagues), and (2) localdelivery of combined immunotherapy to induce an in situ cancer vaccine(P. Sondel and colleagues). As ¹³¹I-NM404 can target cancers ofvirtually any histology, and the local administration of anti-tumormAb+IL2 could potentially be used for virtually any cancer type (sincetumor reactive mAbs are approved or in clinical testing for nearly allcancer histological types), the clinical translation of the combinedstrategy will potentially result in clinically effective therapy forvirtually all high risk cancers.

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Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration from the specification andpractice of the invention disclosed herein. All references cited hereinfor any reason, including all journal citations and U.S./foreign patentsand patent applications, are specifically and entirely incorporatedherein by reference. It is understood that the invention is not confinedto the specific reagents, formulations, reaction conditions, etc.,herein illustrated and described, but embraces such modified formsthereof as come within the scope of the following claims.

The invention claimed is:
 1. A method of treating a metastatic cancer ina subject, wherein the metastatic cancer comprises a primary malignantsolid tumor and one or more metastatic tumors capable of causingconcomitant immune tolerance, the method comprising the steps of: (A)determining an immunomodulatory dose of the radiohalogenated compoundhaving of formula I

or a salt thereof, wherein: R₁ comprises a therapeutic radioactivehalogen isotope; a is 0 or 1; n is an integer from 12 to 30; m is 0 or1; Y is selected from the group consisting of —H, —OH, —COOH, —COOX,—OX, and —OCOX, wherein X is an alkyl or an arylalkyl; and R₂ isselected from the group consisting of N⁺H₃, —N⁺H₂Z, —N⁺HZ₂, and —N⁺Z₃,wherein each Z is independently an alkyl or an aryl, wherein thedetermination of the immunomodulatory dose comprises (i) administeringto the subject a detection-facilitating dose of formula I wherein theradiohalogen isotope is ¹²⁴I, (ii) detecting signals originating fromthe one or more metastatic solid tumors within the subject that arecharacteristic of the ¹²⁴I of formula 1, and (iii) calculating animmunomodulatory dose of radiohalogenated formula 1 for delivery to themetastatic tumors based on the strength of the signals detected in (ii),wherein the radiohalogen of formula 1 is a therapeutic radiohalogen; (B)administering to the subject the calculated immunomodulatory dose ofradiohalogenated formula I that is differentially taken up by andretained within malignant solid tumor tissue; and (C) performing in situtumor vaccination in the subject at the primary malignant solid tumorcomprising contacting the primary malignant solid tumor with acomposition comprising one or more agent capable of stimulating specificimmune cells within the tumor microenvironment whereby the concomitantimmune tolerance caused by the metastatic tumors is prevented and themetastatic cancer is treated.
 2. The method of claim 1, wherein thedetection facilitating dose administered to the subject comprises NM404having the formula of claim 1 wherein R₁ is ¹²⁴I, a is 1, n is 18, m is0 and R₂ is N⁺(CH₃)₃.
 3. A method of treating a metastatic cancer in asubject, wherein the metastatic cancer comprises a primary malignantsolid tumor and one or more metastatic tumors capable of causingconcomitant immune tolerance, the method comprising the steps of: (A)determining an immunomodulatory dose of radioiodinated MIBG, wherein theradioiodine of MIBG is a therapeutic isotope, wherein the determinationof the immunomodulatory dose comprises (i) administering to the subjecta detection-facilitating dose of MIBG wherein the radioiodine isotope is¹²⁴I, (ii) detecting signals originating from the one or more metastaticsolid tumors within the subject that are characteristic of the ¹²⁴I ofMIBG, and (iii) calculating an immunomodulatory dose of radioiodinatedMIBG for delivery to the metastatic tumors based on the strength of thesignals detected in (ii), wherein the radioiodine is a therapeuticisotope; (B) administering to the subject the calculatedimmunomodulatory dose of radioiodinated MIBG that is differentiallytaken up by and retained within malignant solid tumor tissue; and (C)performing in situ tumor vaccination in the subject at the primarymalignant solid tumor comprising contacting the primary malignant solidtumor with a composition comprising one or more agent capable ofstimulating specific immune cells within the tumor microenvironmentwhereby the concomitant immune tolerance caused by the metastatic tumorsis prevented and the metastatic cancer is treated.
 4. The method ofclaim 3, wherein the radioiodine isotope of the immunomodulatory dose isselected from the group consisting of ¹²³I, ¹²⁵I and ¹³¹I.